Patients with acute lung injury are often placed on positive-pressure mechanical ventilation to improve gas exchange. However, mechanical ventilation may generate large shear forces during the cyclic closure and reopening of fluid filled alveoli, while the relatively spared, air-filled alveoli are cyclically overdistended. This may cause or worsen ventilator induced lung injury which may have a negative impact in patients with acute lung injury. In alveolar epithelial cells keratin intermediate filaments (IF) are the major structural proteins. Keratin IF are known to play an important role in maintaining the mechanical integrity of epithelial cells, and in vitro they are able to withstand a wide range of strain conditions without alterations in their structural integrity. In response to shear stress in vivo, IF have been shown to undergo adaptive changes in response to shear stress. This application proposes to study the response of keratin IFs to cyclic stretch, and cyclic and continuous shear stress in alveolar epithelial cells, and determine the effect of these changes in keratin IF on alveolar epithelial function via three interrelated specific aims. Specific aim#1. To determine whether cyclic stretch and/or shear stress-induced changes in keratin IFs alter alveolar fluid reabsorption in wild-type and keratin 8 knockout mice. Specific aim #2. To determine whether cyclic shear stress and/or stretch-induced changes (disassembly) in keratin IFs are mediated by protein kinase C-dependent phosphorylation in alveolar epithelial cells. Specific aim #3. To determine whether cyclic stretch and/or shear stress causes changes in ubiquitination and the regulated degradation of keratin IF networks by the ubiquitin-proteasome pathway in alveolar epithelial cells leading to lung cell injury. Completion of these studies will provide new insights into mechanisms responsible for cyclic stretch-and shear stress-induced alveolar epithelial lung injury.